Deep-Red and Near-Infrared Xanthene Dyes for Rapid Live Cell

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Deep-red and near-infrared xanthene dyes for rapid live cell imaging Guangle Niu, Weimin Liu, Bingjiang Zhou, Hongyan Xiao, Hongyan Zhang, Jiasheng Wu, Jiechao Ge, and Pengfei Wang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00981 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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The Journal of Organic Chemistry

Deep-red and near-infrared xanthene dyes for rapid live cell imaging Guangle Niu,†,‡ Weimin Liu,*,† Bingjiang Zhou,†,§ Hongyan Xiao,† Hongyan Zhang,† Jiasheng Wu,† Jiechao Ge,† and Pengfei Wang† †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials and CityU-CAS Joint

Laboratory of Functional Materials and Devices, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡

§

University of Chinese Academy of Sciences, Beijing, 100049, China

Present Addresses: Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, 100084, China *[email protected]

RECEIVED DATE ( )

ACS Paragon Plus Environment


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Abstract In this work, two xanthene dyes (H-hNR and TF-hNR) have been synthesized by a convenient and efficient method. These two dyes exhibited deep-red and near-infrared (NIR) emissions, high fluorescence quantum yields (QYs), and good photostability. Their structure-optical properties were investigated by X-ray crystal structure analysis and density functional theory calculations. Live cell imaging data revealed that H-hNR and TF-hNR could rapidly stain both A549 and HeLa cells with low concentrations. The excellent photophysical and imaging properties render them as promising candidates for use in live cell imaging. Introduction In the past decades, fluorescence imaging based on small-molecule organic dyes has become a powerful tool to visualize biological events in living systems, such as biomolecule detection1, labeling2, and cancer diagnosis3. In contrast to fluorophores with emissions in short-wavelength regions, deep-red and near-infrared (NIR) fluorophores4 are favored in in vivo bioanalysis and bioimaging due to decreasing light scattering and background autofluorescence, minimizing photodamage to biological samples, and improving tissue depth penetration.5 Xanthenes, mainly including fluoresceins and rhodamines, have been widely used in biological related applications owing to their outstanding photophysical properties.6 However, classical xanthenes exhibit maximum emissions in visible regions which results in low contrast in living systems. Substantial efforts have been made to synthesize xanthene derivatives with longer emission wavelengths. Early attempt to develop long-wavelength xanthene derivatives was introducing electron-withdrawing groups (CF3, perfluorophenyl group, perfluoroalkyl group and CN)7 into the xanthene skeleton at position 9. Later, Lin and others developed several kinds of deep-red and NIR xanthene derivatives with moderate to high fluorescence QYs by extending the π-conjugation of the xanthene skeleton8. Qian and Nagano et al.9


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innovatively proposed that replacing the bridged oxygen atom with other elements (Si, Ge, Sn, Te, or P) could convert traditional xanthenes to other derivatives with narrower energy gaps, resulting in much longer emission wavelengths and good fluorescence QYs. Recently, the hybridization of two classical fluorescent dyes has also become a promising strategy for the design of long-wavelength fluorescent dyes.10 For example, our group reported a family of coumarin-fused pyronin dyes (CP dyes) with deep-red emissions for subcellular organelle imaging.11 However, the incubation concentrations of these CP dyes for imaging were in the range of 5 – 20 μM due to the low fluorescence QYs in Tris–HCl–EDTA (TE) buffer. The lactone group in the skeleton of CP dyes might be the main reason for their low fluorescence QYs, which was probably due to the strong interaction with polar solvent12 and intramolecular charge transfer (ICT)13 in coumarin parts. In addition, the aggregation in polar solvent might also quench the fluorescence due to excellent planarity of CP dyes14. We suspected that replacement the lactone group in the skeleton of CP dyes with ethylene group could inhibit the above-mentioned factors, resulting in high fluorescence QY. In addition, introducing electron-withdrawing group (CF3) could be utilized to develop NIR dye.7b,15 Towards this end, we herein synthesized two xanthene dyes (H-hNR and TF-hNR) through effective condensation reaction, and investigated their photophysical properties, X-ray crystal structure, density functional theory (DFT) calculations, and bioimaging properties in living cells. Results and Discussion Synthesis and Characterization. The synthesis routes of H-hNR and TF-hNR are outlined in Scheme 1. Compound 2 was synthesized through dimethylation of commercial available compound 1 with CH3I and K2CO3 in DMF, and its synthesis was reported by our previous work.16 Thus, H-hNR was synthesized by condensation reaction of compound 2 and commercial compound 6 in concentrated H2SO4 at 100 ℃, with a high yield of 81%. Introducing electron-withdrawing group (CF3) into the xanthene skeleton at position



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9 can be utilized to develop new dyes with emissions in the NIR region.7b,15 Compound 5 was obtained from commercial compound 3 via two steps. First, acetylation of compound 3 in anhydrous CH2Cl2 with Ac2O produced compound 4. Trifluoroacetyl group was introduced into compound 4 via the Friedel-Crafts reaction and then deacetylation by 2N HCl in THF lead to the formation of compound 5. The detailed information of synthesis of compound 5 was reported in our another work.15a Similarily, compound 2 and compound 5 were heated at 100 ℃ in concentrated H2SO4 through condensation reaction produced TF-hNR (16%). The synthesized dyes (H-hNR and TF-hNR) were characterized by 1H NMR, 13

C NMR, 19F NMR and HRMS.

Scheme 1. Synthesis of deep-red and NIR dyes H-hNR and TF-hNR O

O CH3I, K2CO3 N

DMF, 45 °C, 24 h

H2N

1

42%

2 O

Ac2O, NEt3, DMAP

1) TFAA, Et2O, reflux, 3h

OH anhydrous CH2Cl2, rt, 24 h

N

98%

3 O

O R N

2

OAc 2) 2N HCl, THF, rt, 24 h

4

+ N

N

OH R=H, 6 R=CF3, 5

CF3 N

40%, two steps

R

concentrated H2SO4 100°C, 4-12 h then HClO4 81% for H-hNR and 16% for TF-hNR

OH

5

ClO4N

O R=H, H-hNR R=CF3, TF-hNR

N

Furthermore, the structures of H-hNR and TF-hNR were further confirmed by X-ray crystal structure analysis (Figure S1-S3 of the Supporting Information). The single crystals of H-hNR and TF-hNR were obtained by slow evaporation of the mixture solvent (MeOH/CH2Cl2 = 2:1, v/v) at ambient temperature. The details of the X-ray experimental conditions, cell data, and refinement data of H-hNR and TF-hNR


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are summarized in Table S1 and S2 of the Supporting Information. H-hNR formed a monoclinic unit cell and the P121/n1 space group, whereas TF-hNR formed a monoclinic unit cell and the C12/c1 space group. The conjugated C–N bond lengths of H-hNR (1.358 Å and 1.359 Å) and TF-hNR (1.360 Å and 1.352 Å) indicated the delocalization of the positive charge. In addition, the slightly distorted and rigid structures H-hNR and TF-hNR could probably render them highly fluorescent due to reducing aggregation between molecules.14 Photophysical Properties and DFT Calculations. The absorption and fluorescence data of H-hNR and TF-hNR in CH2Cl2, EtOH, and TE buffer are summarized in Table 1, and the corresponding spectra are shown in Figure 1, S5 and S6 of the Supporting Information. The maximum absorption wavelengths (λabsmax) of H-hNR in different solvents were in the visible light region, whereas the maximum fluorescence wavelengths (λemmax) were mainly in the deep-red region. Remarkably, H-hNR exhibited high QYs, particularly in CH2Cl2 and EtOH. Compared with previously reported dye CP11a (structure shown in Figure 2), H-hNR exhibited much higher quantum yield (Table 1). The replacement of the lactone group with ethylene group might weaken the interaction with solvent, thus resulting in reduced non-radiative transitions.12 Additionally, reduced ICT effect could also attribute to the high quantum yield of H-hNR due to the smaller Stokes shift of H-hNR in comparison with that of CP.13 Previous studies have proved that the introduction of electron-withdrawing group CF3 at position 9 of xanthenes leads to dyes with longer absorption and emission, because the electron-withdrawing group CF3 could largely decrease the energy of excited state, resulting in narrower bad gap and red-shifted absorption and emission.7b,15 Compared with the absorption and fluorescence wavelengths of H-hNR, those of TF-hNR were extended to the deep-red and NIR regions, respectively. The QY of TF-hNR was high in CH2Cl2 but was sharply reduced from CH2Cl2 and EtOH to TE buffer because of its very narrow energy gap and enhanced non-radiative transitions in NIR fluorescent dyes.17



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Table 1. Photophysical Properties of H-hNR, TF-hNR and CP dye

H-hNR

TF-hNR

CP11a

solvent

λabsmax/λemmax (nm)

Stokes shift (cm-1)

ε (M cm-1)

Φa/Φb

CH2Cl2

619/642

579

133200

0.677/0.724

EtOH

612/644

812

78200

0.576/0.618

TE buffer

604/641

956

65500

0.310/0.282

CH2Cl2

684/713

595

97100

0.310/0.336

EtOH

680/725

913

54400

0.119/0.163

TE buffer

672/716

914

47100

0.034/0.039

CH2Cl2

618/648

749

80800

0.220/0.265

TE buffer

598/658

1524

39000

0.010/0.009

-1

Conditions: Φa is the relative fluorescence quantum yield determined by using cresyl violet (ref. 21) and zinc phthalocyanine (ref. 22) as the standards; Φb is absolute fluorescence quantum yield measured by using an integrating sphere.

Figure 1. Normalized absorption and fluorescence spectra of H-hNR and TF-hNR in TE buffer.


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Figure 2. The molecular structure of CP11a. To further investigate the optical properties of H-hNR, TF-hNR and CP, we performed DFT calculations at the B3LYP/6-31G level of theory by using the Gaussian 09 program package.18 The calculated HOMOs and LUMOs of H-hNR, TF-hNR and CP are shown in Figure 3 and the optimized structures are shown in Figure S7 of the Supporting Information. Compared with CP, H-hNR exhibited slightly higher HOMO and LUMO energy levels probably due to reduced ICT effect while the energy gap was comparable to that of CP. In addition, the electron density of LUMOs was not localized in the ethylene part of H-hNR while that was localized in the lactone part of CP, which further indicated the decreased ICT effect for H-hNR. As for TF-hNR, the HOMO and LUMO energy levels were decreased due to the electron-withdrawing group CF3, thus results in narrower energy gap and red-shifted absorption and emission, which was in good consistent with measured photophysical data (Table 1). The optimized the structures (Figure S7 of the Supporting Information) further showed that H-hNR and TF-hNR exhibited twisted molecular structures while CP exhibited planar structure, which probably had an important effect on the emissions of these dyes.



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Figure 3. Spatial distributions of calculated HOMOs and LUMOs and HOMO/LUMO energy gaps (unit: eV) of H-hNR, TF-hNR and CP. The effects of pH on the absorption spectra of H-hNR and TF-hNR were investigated in different Britton–Robinson (BR) buffers (40 mM, pH 2–11). The corresponding data are shown in the Figure 4a and Figure S8. The absorption intensity of H-hNR varied slightly within the pH range of 2–8 but dropped sharply beginning from the pH value of 9. Such decrease is attributed to the attachment of hydroxyl under highly alkaline environment conditions. For TF-hNR, the absorption intensity remained stable mainly under acidic conditions (pH 2–6) but dropped significantly when the pH value was higher than 7 as a result of the easier attachment of hydroxyl under alkaline environment conditions, which resulted from the high electron deficiency of the position directly connected to CF3 of TF-hNR. The data on the effect of pH on TF-hNR is consistent with our previously reported data on AFR3 (Figure 4b).15a


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Figure 4. (a) pH-dependence of the normalized absorption of H-hNR and TF-hNR. (b) The molecular structure of AFR315a. Considering that reduced glutathione (GSH) with high levels is present in live cells and GSH could react with reactive fluorophores, we texted the the stability of H-hNR and TF-hNR in the presence of 10 mM GSH. After addition of 10 mM GSH for 10 min, the absorptions of H-hNR and TF-hNR were measured and the corresponding data was summarized in Figure S9 of the Supporting Information. We could clearly see that the absorption intensity of H-hNR showed very little change after adding of 10 mM GSH. For TF-hNR, the absorption intensity slightly increased after adding of 10 mM GSH. These data further demonstrate that H-hNR and TF-hNR exhibit good stability in the presence of high levels of GSH. Bioimaging and Cell Location. The cell uptake of H-hNR and TF-hNR was studied in A549 cells and HeLa cells by confocal laser scanning microscopy. We first performed cell-imaging experiments with different concentrations of H-hNR and TF-hNR (Figure 5). After incubation for 10 min, clear fluorescence of H-hNR in the nucleolus and cytoplasm can be detected at a concentration as low as 200 nM. For TF-hNR, an optimized concentration at 2 μM was adopted to obtain clear fluorescence in the nucleolus and cytoplasm. In addition, cell imaging experiments at different time-points were also conducted, and the corresponding imaging of H-hNR and TF-hNR was obtained (Figure 6 and Figure S10). For most of the fluorophores staining nucleolus, long incubation time (tens of minutes or even several hours) is needed to obtain satisfactory images of nucleolus imaging in live cells.19 Parker et al.19b synthesized a europium complex staining nucleolus in live cells with at least 1 h. Tang’s group reported a α-cyanostilbene derivative (ASCP) for dual-colour nucleolus and mitochondria imaging with up to 30-min incubation.19h Compared with these fluorophores, H-hNR and TF-hNR could easily enter live cells with rapid localization. The fluorescence intensities in the nucleolus and cytoplasm almost reached saturation in



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approximately 9 min for both H-hNR and TF-hNR, indicating that they had excellent cell plasma and nuclear membrane permeability.

Figure 5. Confocal fluorescence microscopy images of HeLa cells stainted with different concentrations of (a) H-hNR and (b) TF-hNR. Scale bar: 10 μm.

Figure 6. Confocal fluorescence microscopy images of HeLa cells stainted with (a) H-hNR (0.8 μM) and (b) TF-hNR (2 μM) at different time-points. Scale bar: 10 μm. DNase and RNase digest experiments were performed in fixed A549 cells to confirm the localization of fluorescence in the nucleolus. As shown in Figure 7, in the presence of DNase, the fluorescence intensities of H-hNR and TF-hNR in the nucleoli and cytoplasm almost remained same, whereas the fluorescence intensity in the nucleolus slightly decreased. By contrast, upon treatment with RNase, the fluorescence intensity in nucleoli significantly dropped, whereas the fluorescence intensity in the cytoplasm was


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slightly enhanced; SYTO@ RNASelectTM showed almost the same tendency in the digest experiments. The fluorescence in the nucleolus almost vanished for DAPI in the presence of DNase, whereas the fluorescence basically remained the same upon treatment with RNase. These imaging data demonstrated that H-hNR and TF-hNR stain nucleolus via the non-covalent interaction with nucleolar RNA.

Figure 7. The DNase and RNase digest experiments. Scale bar: 10 μm. For the fluorescence obtained in cytoplasm, two commercial dyes, MitoTracker Green FM (MTG) and LysoTracker Green DND-26 (LTG), were used for co-staining with H-hNR or TF-hNR to confirm the localization. Colocalization imaging data (Figure 8, Figure S11 and Table S3) showed that H-hNR mainly located in the mitochondria with good overlap with MTG, while the fluorescence observed for TF-hNR in cytoplasm was mainly associated with lysosomes. Notably, the fluorescence of TF-hNR in lysosomes was brighter than that in nucleolus because TF-hNR showed strong emission under acidic conditions (Figure 4a) and lysosomes are acidic organelles (pH 4.6–5.0)20.



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Figure 8. Confocal fluorescence microscopy images of A549 & HeLa cells (a) stained with MTG (100 nM) and H-hNR (0.8 μM) and (b) stained with LTG (200 nM) and TF-hNR (2 μM). Scale bar: 10 μm. Photostability and Cytotoxicity. The photostability and cytotoxicity of H-hNR and TF-hNR are crucial indicators of synthesized dyes for imaging-related biological applications. The photostability of H-hNR and TF-hNR was evaluated in HeLa cells fixed in prechilled methanol by continuous irradiation with laser. Figure 9 showed that the intensity of H-hNR and TF-hNR decreased to 90% after 30 min of continuous irradiation. As a comparison, however, over 90% of the initial fluorescence intensity of commercial SYTO@ RNASelectTM was significantly decreased only in 10 min, and the intensity of MTG and LTG decreased to 6.4% and 51.2% after 30 min of continuous irradiation, respectively. As for evaluating

the

cytotoxicity

of

H-hNR

and

TF-hNR,

standard

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium) assays were performed in A549 and HeLa cells. After incubation in HeLa cells for 24 h, the data showed that H-hNR and TF-hNR exhibited negligible cytotoxicity at low concentrations (Figure S12 of the Supporting Information).


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Figure 9. Photostability of H-hNR, TF-hNR, SYTO@ RNASelectTM, MTG and LTG in HeLa cells fixed with prechilled methanol. Irradiation conditions: For H-hNR, 640 nm laser, laser power 8%；For TF-hNR, 640 nm laser, laser power 40%；For SYTO@ RNASelectTM, 488 nm laser, laser power 8%；For MTG, 488 nm laser, laser power 8%；For LTG, 488 nm laser, laser power 8%. Conclusions In summary, we successfully synthesized two xanthene dyes (i.e., H-hNR and TF-hNR) with deep-red and NIR emissions by facile and straightforward methods. The X-ray diffraction structures, DFT calculations, and photophysical properties of H-hNR demonstrated that the ethylene group in the skeleton can efficiently improve the QYs in polar solution. Cell imaging studies reveal that H-hNR and TF-hNR located in nucleolus and mitochondria (or lysosomes) in both A549 and HeLa cells with short incubation time and low concentration. Moreover, these two dyes show excellent photostability and low cytotoxicity. This work may provide an opportunity for the construction of other xanthene dyes with high QYs for live cell imaging. Experimental Section General All commercial chemicals were used as received without further purification. SYTO@



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RNASelectTM, DAPI, MitoTracker Green FM (MTG) and LysoTracker Green DND-26 (LTG) were purchased from Invitrogen (USA). DNase and DNase-free RNase were purchased from Takara. 1H NMR (400 MHz), 13C NMR (100 MHz) and 19F NMR (376 MHz) spectra were recorded on a 400, 100 and 376 MHz instrument, respectively, with tetramethylsilane as an internal standard. Electrospray ionization high resolution mass spectra (ESI-HRMS) were measured with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS). All absorption and fluorescence spectra in this work were obtained at room temperature, respectively. Relative fluorescence quantum yield (QY) measurement. Cresyl violet (CV) in methanol (Φ = 0.54)21 and zinc phthalocyanine (Zp) in toluene (containing 1% pyridine; Φ = 0.30)22 were chosen as the standards for H-hNR and TF-hNR, respectively. The fluorescence quantum yields of H-hNR and TF-hNR were calculated according to the following equation: Φx=Φst*[nx2/nst2]*[Gx/Gst], where Φ is the fluorescence quantum yield, G is the gradient from the plot of integrated fluorescence intensity versus absorbance, n is the refractive index of different solvents. The subscript “st” and “x” refer to the standard and unknown and dye, respectively. The excitation wavelengths (λex) for QY measurement of H-hNR and TF-hNR were 540 nm and 605 nm, respectively. And the absorbance values of all dyes were below 0.08. The related data are shown in Figure S4 of the Supporting Information. Imaging. Two tumour cell lines, A549 (human lung adenocarcinoma epithelial cell line), and HeLa (human cervical adenocarcinoma epithelial cell line) were used to perform the imaging-related experiments. A549 and HeLa cells were cultured in McCoy's 5A and Dulbecco’s modified Eagle medium (DMEM) respectively, containing 10% fetal bovine serum (FBS) including 1% antibiotics (50 unit/mL penicillin, and 50 mg/mL of streptomycin) under 5% CO2 at 37 °C in a humidified incubator. After incubation for 24 h, fresh culture media (containing 0.8 μM H-hNR and 2 μM TF-hNR) were added into the confocal dishes, the confocal dishes were incubated for 10 min prior to conduct the experiments. As


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for colocalization experiments, apart from the above-mentioned concentrations of H-hNR and TF-hNR in the fresh culture media, commercial available nucleolar RNA dye SYTO@ RNASelectTM (1 μM), mitochondria-specific green fluorescent dye MitoTracker Green FM (MTG, 100 nM) and lysosome-specific green fluorescent dye LysoTracker Green DND-26 (LTG, 200 nM) were also contained to perform the corresponding imaging experiments. All the confocal dishes were washed with PBS (phosphate-buffered saline) before imaging. Fluorescent images were acquired with a confocal laser scanning microscopy (foe DAPI, excited at 405 nm, DAPI channel; for SYTO@ RNASelectTM, MTG and LTG, excited at 488 nm, FITC channel; for H-hNR and TF-hNR, excited at 640 nm, Cy5 channel.). Images were processed by NIS-Elements Viewer 3.20. For photostability, SYTO@ RNASelectTM, MTG and LTG were continuous irradiated with a 488 nm laser, and a 640 nm laser was used for H-hNR and TF-hNR. DNase and RNase digest experiments. A549 cells were fixed in prechilled methanol at −20 °C for 1 min, then the cells was washed with l mL PBS with 1% Triton X-100 for 1 min at room temperature to permeabilize the cell membrane. After washing with PBS twice, 1 mL PBS (containing 0.8 μM H-hNR, 2 μM TF-hNR, 1 μg/mL DAPI and 0.5 μM SYTO@ RNASelectTM) solution were added. Then incubation for 20 min at room temperature, they were rised clean by PBS twice. A total of 1 mL PBS (as the control experiment), 1 mL DNase solution (20 u/mL in PBS), and 1 mL DNase-free RNase solution (25 μg/mL in PBS) were added into the three other dishes. After incubation for 2 h under 5% CO2 at 37 °C in a humidified incubator, the cells were rinsed clean by PBS twice more before imaging. To obtain the fluorescent imaging pictures under the same condition, equal exposure time and laser intensity were used for the control, DNase, and RNase experiments. Synthesis of H-hNR. The compound H-hNR was synthesized following our previous method.15a Concentrated H2SO4 (5 mL) was initially poured into a round-bottomed flask, and the solution was stirred



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at room temperature. Then compounds 2 (189 mg, 1.0 mmol) and 6 (193 mg, 1.0 mmol) were successively added, and the mixture was heated at 100 °C for 4 h. After cooling to room temperature, the mixture was poured into ice water. Perchloric acid (1 mL) was slowly added into the solution, and the resulting precipitate was filtered and washed with generous amounts of water. The sample was dried and purified by silica gel chromatography using v(CH2Cl2)/v(CH3OH) = 30:1 as the eluent to obtain green solid H-hNR (360 mg, 81%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.35 (s, 1H), 8.09 (d, J = 9.1 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.22 (dd, J = 9.2, 2.1 Hz, 1H), 7.16 (s, 1H), 6.90 (d, J = 9.2 Hz, 1H), 6.77 (s, 1H), 3.61 (q, J = 7.0 Hz, 4H), 3.19 (s, 6H), 2.98 (s, 4H), 1.22 (t, J = 7.0 Hz, 6H).

13

C NMR (100 MHz, DMSO-d6): δ

(ppm) 164.5, 156.6, 155.0, 153.4, 145.6, 144.6, 130.7, 128.9, 120.2, 115.1, 114.9, 112.8, 111.9, 110.8, 95.8, 44.8, 39.9 (overlapped with DMSO-d6), 27.1, 25.1, 12.4. HRMS (ESI) calcd for [C23H27N2O]+ m/z 347.21179 [M]+, found m/z 347.21180. Synthesis of TF-hNR. The compound TF-hNR was synthesized following our previous method.15a To a Concentrated H2SO4 (10 mL) was initially poured into a round-bottomed flask, and the solution was stirred at room temperature. Then compounds 2 (378 mg, 2.0 mmol) and 5 (522 mg, 2.0 mmol) were successively added, and the mixture was heated at 100 °C for 12 h. After cooling to room temperature, the mixture was poured into ice water. Perchloric acid (2 mL) was slowly added into the solution, and the resulting precipitate was filtered and washed with generous amounts of water. The sample was dried and purified by silica gel chromatography using v(CH2Cl2)/v(CH3OH) = 70:1 as the eluent to obtain green solid TF-hNR (164 mg, 16%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.20 (d, J = 9.3 Hz, 1H), 7.81 (dd, J = 9.4, 2.1 Hz, 1H), 7.24 (dt, J = 9.5, 2.6 Hz, 2H), 7.00 (dd, J = 9.3, 2.5 Hz, 1H), 6.84 (d, J = 2.3 Hz, 1H), 3.61 (q, J = 7.0 Hz, 4H), 3.28 (s, 6H), 3.11 (t, J = 6.5 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H), 1.22 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 164.9, 156.1, 155.9, 152.4, 146.7, 135.7 (q, J = 303 Hz), 130.6, 126.5 (q, J = 5 Hz), 124.5, 121.7, 120.5 (q, J = 3 Hz), 115.4, 113.4 (q, J = 5 Hz), 110.6, 109.2, 96.7,


Page 17 of 20

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44.8, 40.3, 26.5, 23.6 (q, J = 4 Hz), 12.5. 19F NMR (376 MHz, DMSO-d6): δ (ppm) –54.0 (s, 3F). HRMS (ESI) calcd for [C24H26F3N2O]+ m/z 415.19917 [M]+, found m/z 415.19917. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grant Nos. 21373250 and 61227008) and 973 Program (No. 2014CB932600). All calculations were performed on the cluster of Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, China. Supporting Information Available: X-ray crystallographic data in CIF format; photophysical data; cell data; the details of DFT calculations (absolute energy and atomic coordinates); NMR spectra of new compounds; HR-MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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